Mycobacterial infection: Immune evasion, host susceptibility and immunological markers of diagnostic importance

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Doctoral thesis from the Department of Immunology,

The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

Mycobacterial infection: Immune evasion, host

susceptibility and immunological markers of

diagnostic importance

John Arko-Mensah

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All previously published papers were reproduced with permission from the publishers.

Printed in Sweden by Universitetsservice AB, Stockholm 2008 Distributor: Stockholm University Library

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SUMMARY

According to the WHO, the interaction between the twin pandemics of human immunodeficiency virus (HIV) and tuberculosis (TB) could soon become a "threat to global health security," particularly with the emergence of almost untreatable strains of Mycobacterium tuberculosis. Understanding the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to

susceptibility of the host to infection may help in the rational design of more effective drugs and vaccines, as well as the development of better diagnostic techniques.

Toll-like receptors (TLRs) are key sensors of microbial infections, and an important link between innate cellular responses and the subsequent activation of adaptive immune defenses against the invading pathogen. A role for persistent TLR2 signalling as an immune evasive mechanism by mycobacteria in the host has been reported. In the first study, we investigated the functional implications of prolonged TLR signalling on interferon-gamma (IFN-γ) mediated killing of mycobacteria by murine macrophages in vitro. Continuous TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither tumor necrosis factor (TNF) nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ.

Receptor mediated recognition and phagocytosis of mycobacteria culminates in a cascade of immunological events, resulting in the production of chemokines and pro-inflammatory cytokines by innate cells, and the subsequent generation of mycobacteria-specific T- and B-lymphocytes, capable of producing soluble mediators of adaptive immunity. In the second paper, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not in serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response.

Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages (BMM) in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria.

In conclusion, the work presented in this thesis provide further insight into the immune evasive mechanisms utilized by mycobacteria to persist in the host, and strengthen the notion that in TB, the nature and level of immune responses in the respiratory tract is more reflective of disease activity than systemic responses. Furthermore, it provides some immunological basis underlying the differences in host susceptibility to mycobacterial infections.

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“It is not the strongest of the species that survives, or the most intelligent that survives. It

is the one that is the most adaptable to change”

Charles Darwin

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ORIGINAL ARTICLES

This doctoral thesis is based on the following papers, which are referred to by their Roman numerals in the text:

I. Arko-Mensah J, Julián E, Singh M, Fernández C. TLR2 but not TLR4 signalling is critically involved in the inhibition of IFN-γ induced killing of mycobacteria by murine macrophages. (Scand J Immunol 2007; 65:148-157).

II. Arko-Mensah J*, Rahman J M*, Julián E, Horner G, Singh M, Fernández C. Increased levels of immunological markers in the respiratory tract but not in serum correlate with active pulmonary mycobacterial infection in mice. Accepted.

III. Arko-Mensah J*, Rahman J M*, Fernández C. Early immune responses are responsible for the better control of pulmonary mycobacterial infection in C57BL/6 compared with BALB/c mice. Manuscript.

*

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ABBREVIATIONS

Ag85 Antigen 85 complex

APC Antigen-presenting cell BAL Broncho-alveolar lavage

BCG Mycobacterium bovis Bacillus Calmette-Guérin BMM Bone-marrow macrophages

CFP Culture filtrate protein CIITA Class II transactivator CR Complement receptor CTL Cytotoxic-T lymphocyte CWBCG BCG cell wall

CWM.vaccae M. vaccae cell wall

DC Dendritic cell

DC-SIGN DC-specific intercellular adhesion molecule-3-grabbing nonintegrin DOTS Directly observed treatment-short course

ESAT-6 Early secretory antigenic target 6 HIV Human immunodeficiency virus hk-BCG Heat killed BCG

HLA Human leukocyte antigen IGRA Interferon-gamma release assay i.n. Intranasal

i.m. Intramuscular

IRAK Interleukin-1-receptor associated kinase i.v. Intravenous IFN-γ Interferon-gamma IL Interleukin LAM Lipoarabinomannan LN Lymph node LPS Lipopolysaccharide MDR Multidrug-resistant MHC Major-histocompatibility complex MyD88 Myeloid differentiation factor 88 MOTT Mycobacteria other than tuberculosis NF-kB Nuclear factor kappa-B

NK Natural killer NK-T Natural killer-T cells

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NO Nitric oxide

NOD Nucleotide-binding oligomerization domain NOS Nitric oxide synthase

Nramp1 Natural resistance associated macrophage protein 1 PAMP Pathogen-associated molecular pattern

PPD Purified protein derivative PRR Pattern recognition receptor RNI Reactive-nitrogen intermediate ROI Reactive-oxygen intermediate s.c. Subcutanous

sst1 Super-susceptibility to tuberculosis 1 sTNFR Soluble tumor necrosis factor receptor TACE Tumor necrosis factor converting enzyme TACO Tryptophan aspartate rich coat protein TB Tuberculosis

TCR T-cell receptor

TGF-β Transforming growth factor-beta TLR Toll-like receptor

TmTNF Transmembrane tumor necrosis factor TNF Tumor necrosis factor

TRAF TNF receptor associated factor TST Tuberculin skin test

WHO World Health Organization WT Wild type

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INTRODUCTION

TUBERCULOSIS

Tuberculosis (TB), also known as the 'white plague'[1], and human immunodeficiency virus (HIV) are the major infectious killers of adults in the developing world, and about 13 million people are infected with these two pathogens. The global epidemic of TB results in 8-10 millionnew cases every year [2], with an annual projected increase rateof 3%. It is estimated that between 5 and 10% of immunocompetent individuals are susceptible to TB, of which, 85% developpulmonary disease [3]. In 1993 and also 2002, the World Health Organization (WHO) declared TB a global public health emergency. The resurgence in the incidence of TB in the last two decades has been attributed to the emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis [4, 5], the causative organism of TB, coinfection with HIV [6, 7], as well as immigration of infected persons from TB prevalent to less prevalent areas.

The genus Mycobacterium comprises mostly soil dwelling saprophytes, and only a few members of the genus have evolved to adopt a pathogenic lifestyle, causing diseases of diverse nature and varying severity [8]. Tuberculosis is caused by members of the M. tuberculosis complex that consists of M. tuberculosis, M. bovis, M. africanum, M. canettii and M. microti. The mycobacteria grouped in the complex are characterized by 99.9% similarity at the nucleotide level and identical 16S rRNA sequences [9, 10], but differ widely in terms of their host tropisms, phenotypes, and pathogenicity. Some are exclusively human pathogens (M. tuberculosis, M. africanum, M. canettii) or rodent M. microti whereas M. bovis have a wide host spectrum [8]. All members of the complex are slow-growing, with generation time ranging from 12 to 24 hrs depending on environmental and microbial variables.

MYCOBACTERIAL INFECTIONS

M. tuberculosis is an obligate, aerobic, intracellular pathogen, which has a predilection for the lung tissue rich in oxygen. TB occurs almost exclusively from inhalation of aerosol droplet

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containing M. tuberculosis expelled by an individual with active pulmonary TB through coughing, spitting, singing and other forced respiratory maneuvers. Usually, repeated exposure to a TB patient is necessary for infection to take place. Inhaled droplets are deposited in the alveolar spaces, where the bacteria are taken up by phagocytic cells, mainly alveolar macrophages [11], an event which induces a rapid inflammatory response and accumulation of cells.

Pathogenesis of TB

Infection with M. tuberculosis may lead to different clinical outcomes [12]:

Primary TB: Clinical symptoms develop within the first 1–2 years of infection, and represent the majority of pediatric cases. Alternatively, infection can lead to chronic, slowly progressive TB in which clinical symptoms develop after more than 2 years of infection. Finally, in 90% of cases, the infection remains latent and totally asymptomatic. The latter two groups of infected individuals constitute the reservoir of M. tuberculosis.

Secondary TB: Seen mostly in adults as a reactivation of previous infection (latent TB), or reinfection, particularly when ones’ health status declines. Typically, the upper lung lobes are most affected, and cavitation can occur.

Dissemination of tuberculosis outside the lungs (extrapulmonary TB) is more common in children and HIV infected individuals [13], leading to the appearance of a number of uncommon findings with characteristic patterns [reviewed in 14]: skeletal TB, involves mainly the thoracic and lumbar vertebrae also known as Pott's disease, genital tract TB involves the fallopian tube, prostate and epididymis. Others are: urinary tract TB, TB of the central nervous system, cardiac TB and scrofula (lymphadenitis TB) [15].

IMMUNE EVASIVE MECHANISMS

M. tuberculosis invades and replicates in macrophages, cells of the host innate defense system designed to eliminate pathogenic microorganisms, through a variety of immune evasion strategies. The use of non-activating complement receptors (CR) to enter into macrophages may be advantageous for the bacterium, since engagement of these receptors does not induce

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the release of cytotoxic reactive oxygen intermediates (ROI) [16]. The ability of pathogenic mycobacteria to adapt to the hostile environment of macrophages has been instrumental in its success as a pathogen. Mycobacteria interfere with host trafficking pathways by modulating events in the endosomal/phagosomal maturation pathway to create a protective niche, the phagosome [17]. The mycobacteria containing phagosome, while connected to the endocytic pathway, does not fuse with lysosomes or mature into phagolysosomes [18, 19]. By blocking its delivery to lysosomes, the mycobacterium is able to avoid the acidic proteases of lysosomes; avoid exposure to the bactericidal mechanisms within lysosomes; prevent degradation and hence processing and presentation of mycobacterial antigens to the immune system [20]. Another mechanism by which mycobacteria could interfere with phagolysosomal fusion is by retention of an important host protein termed (tryptophan aspartate containing Coat protein (TACO), also known as coronin 1 on the phagosome [18], thereby behaving as self antigens. TACO represents a component of the phagosome coat, and retention of TACO prevents phagosomes from fusing with lysosomes, thereby contributing to the long-term survival of bacilli within the phagosome.

The recognition of infected macrophages by CD4+ T cells depends on constitutively expressed major histocompatibility complex (MHC) class II on professional antigen-presenting cells (APCs), level of which is upregulated upon activation with IFN-γ. One mechanism by which M. tuberculosis avoids elimination by the immune system after infection is through the inhibition of MHC II expression or antigen processing or presentation by macrophages [21-24]. Inhibition of MHC II expression or antigen processing does not require viable bacilli and can be achieved by exposure to bacterial lysate [21, 22, 25]. The M. tuberculosis 19-kDa lipoprotein (19-kDa) was identified as the predominant ligand involved in inhibiting MHC II expression and antigen processing in a toll-like receptor (TLR) 2 dependent manner [26]. Subsequently, several studies have shown that 19-kDa inhibits the expression of several interferon gamma (IFN-γ) responsive genes, including MHC class II transactivator (CIITA) and MHC II, as well as class II dependent antigen presentation in a TLR2 dependent manner [27-29]. Moreover, we (paper I), and others [30] have demonstrated that signalling through TLR2 by 19-kDa inhibits IFN-γ -mediated killing of ingested mycobacteria by murine macrophages.

It was recently demonstrated that M. tuberculosis uses at least two mechanisms to block responses to IFN-γ; one initiated by lipoproteins acting through TLR2/ MyD88 (myeloid

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differentiation factor 8), whereas the other is initiated by mycobacterial peptidoglycan (PGN), acting in a TLR2-, MyD88-independent manner [30]. Other immune evasive mechanisms include the secretion of enzymes such as superoxide dismutase or catalases by M. tuberculosis, which are antagonistic to ROI [31], or the inhibition of macrophage apoptosis [32]. Furthermore, macrophages infected with M. tuberculosis produce inhibitory cytokines, such as transforming growth factor (TGF)-β and interleukin (IL)-10, which reduce macrophage activation, thereby leading to decreased clearance of bacteria [33, 34].

IMMUNITY TO MYCOBACTERIAL INFECTIONS

Innate immunity

It is believed that the host innate immunity provides the initial resistance to infections with intracellular pathogens, such as mycobacteria, before the adaptive type 1 cell-mediated immunity fully develops. The major cellular components involved in innate immunity include phagocytes; macrophages, neutrophils, dendritic cells (DCs); natural killer (NK) cells; γδ T cells, and soluble mediators released by these cells serve as a linker to cell-mediated immunity. During the initial phase of infection, mycobacteria are ingested by resident alveolar macrophages. However, mycobacteria can also be ingested by alveolar epithelial type II pneumocytes [35], found in greater numbers than macrophages in alveoli.

Overall, phagocytic cells play a key role in restricting the multiplication and dissemination of intracellular pathogens, as well as initiation and direction of the adaptive immune response. In addition, DCs, known to be much better antigen presenters than macrophages [36, 37], play an important role in the early stages of infection through presentation of specific mycobacterial antigens to T cells [38]. A number of receptors are critical for M. tuberculosis detection and uptake by phagocytes.

Receptor mediated detection of mycobacteria

Entry of mycobacteria into phagocytic cells can occur through binding to multiple receptors. In human macrophages, the primary receptors for M. tuberculosis recognition and uptake are the mannose receptors and complement receptors 3 [39, 40].

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Other receptors have been shown to interact with mycobacteria: surfactant protein A and its receptors, scavenger receptor class A, mannose binding lectin, and possibly dectin-1 [reviewed in 41, 42]. The mode of entry into macrophages is considered as predetermining the subsequent intracellular fate of mycobacteria. In contrast to human macrophages, human DCs primarily use DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) for mycobacterial detection and uptake, with no significant role for complement or mannose receptors [43]. However, experiments have shown that blocking individual receptors does not significantly alter M. tuberculosis intracellular trafficking [41].

Toll-like receptors

One of the earliest indications that the body has been infected with an invading microbe is the activation of signalling pathways upon recognition of specific components conserved among microorganisms, known as pathogen-associated molecular patterns (PAMPs) by evolutionarily ancient germline-encoded receptors, the pattern recognition receptors (PRRs) [44]. The most studied PRRs, the TLRs constitute a family of transmembrane proteins expressed on many cells, including cells of the innate immune system such as macrophages and DC. Messenger RNA expression for TLRs 1-9 has been shown in human lungs, indicating that it is a major site for TLR activity [45]. This is important since lungs are the primary target for infection by many pathogens including M. tuberculosis.

Some of the bacterial molecules that are recognized by TLRs include lipopeptides by TLR2 (as a heterodimer with TLR1 or TLR6), lipopolysaccharide (LPS) by TLR4, flagellin by TLR5, and bacterial CpG DNA by TLR9. TLRs, with the exception of TLR3, require the adaptor molecule MyD88 for signal transduction (Figure 1) [46].

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Figure 1. Microbial ligands and association with known TLRs and adaptor molecules. (Microbes Infect 2004; 6:946-959). Reprinted with permission from Elsevier.

TLR signal transduction is mediated by binding of the intracellular adaptor protein MyD88 to the TIR domain of TLRs, followed by the recruitment of IL-1 receptor associated kinases (IRAK), tumor necrosis factor (TNF) receptor associated factor (TRAF), mitogen-activated protein kinase (MAPK) leading to translocation of nuclear factor-κappa B (NF-κB) [46, 47]. Translocation of the cytoplasmic factor NF-κB results in the transcription of several genes, leading to activation of pro-inflammatory and antibacterial effector pathways, which include production of pro-inflammatory cytokines such as TNF and ILs, chemokines, nitric oxide (NO) and defensins [48, 49]. TLR signalling also triggers differentiation of monocytes into macrophages and DCs, thereby generating the cellular populations necessary for a potent innate and adaptive immune response [50]. In human naive B cells, TLRs are expressed at low to undetectable levels, but their expression is rapidly up-regulated upon B-cell receptor

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trigerring by microbial PAMPs [51]. In contrast however, memory B cells express several TLRs at consitutively high levels. Although B cell-intrinsic TLR signalling is not required for antibody production, it plays a role in the amplification of the humoral immune response [52]. Therefore, in addition to their role in innate immunity, TLRs are also critically involved in the initiation and enhancement of adaptive immune responses [49].

In experimental TB, TLR2-mediated signalling of APCs by mycobacterial components is the most studied. It was shown that direct activation of TLR2 by the 19-kDa resulted in reduced viability of ingested bacilli in human macrophages in a TNF and NO independent manner [53]. Moreover, TLR2 signalling can also promote apoptosis of M. tuberculosis infected macrophages [54]. Other signals also contribute to the pro-inflammatory response; TLR-1/TLR6 and TLR4 have been implicated in responses to M. tuberculosis [55]. In vitro, mice deficient in TLR2 [56], or the TLR adaptor molecule MyD88 displayed a higher susceptibility to mycobacterial infection [57]. It has also been shown that nucleotide-binding oligomerization domain (NOD) 2 is a nonredundant PRR of mycobacteria, and synergizes with TLRs in the stimulation of cytokine production by phagocytic cells [58, 59]. Furthermore, mannose-capped lipoarabinomannan (LAM), a component of M. tuberculosis cell wall, can deliver anti-inflammatory signals through DC-SIGN on DCs, thereby reducing antimycobacterial activity and stimulating the release of IL-10 [43].

Macrophages

A key characteristic of M. tuberculosis infection is that this bacterium multiplies intracellularly, primarily in macrophages, evading in this way many host-defense mechanisms [60]. Thus, internalization of the bacterium by alveolar macrophages is a critical step for the establishment of TB infection. In the lung, the bacteria are phagocytosed by alveolar macrophages and induce a localized pro-inflammatory response and rapid production of cytokines such as TNF, IL-1 and IL-6, and chemokines that lead to the recruitment of immune cells to the site of infection [61-63]. After internalization, macrophages process and present antigens on both MHC I and II to T cells, which in turn secrete IFN-γ, required for the induction of antimycobacterial activity.

A major effector mechanism responsible for the antimycobacterial activity of IFN-γ and TNF is the induction of NO and related reactive nitrogen intermediates (RNIs) by macrophages via

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the action of inducible form of nitric oxide synthase (NOS) [61, 64]. Whereas the antimycobacterial property of RNI is well documented both in vitro and in vivo in the murine model [64-66], there has been conflicting data on the role of RNI in human TB. However, recent data support a protective role for these reactive intermediates in human TB as well [67]. Other antimycobacterial mechanisms of macrophages are: phagolysosome fusion, a process which exposes ingested bacteria in the phagosome to lytic enzymes in the lysosome [18, 20]; apoptosis of infected macrophages [54], which removes the niche for growth and therefore restricts multiplication of bacteria. Moreover, recent studies have demonstrated that autophagy; the cellular process by which a cell degrades its own intracellular compartments is a previously unappreciated innate immune defense mechanism [68]. Stimulation of mouse macrophages with IFN-γ induced autophagy which was necessary for antimicrobial activity against M. tuberculosis. A separate study demonstrated that lysosomal hydrolyzed ubiquitin peptides have direct antimicrobial activity against M. tuberculosis, and are delivered in an autophagy dependent manner to phagosomes harbouring mycobacterium [69].

During M. tuberculosis infection, macrophages and their circulating precursors present at or recruited to the site of infection phagocytose bacteria and migrate deeper into lung tissues, thereby playing important roles in immune activation and bacteria dissemination [70]. The zebrafish embryo infection model by M. marinum has helped to elucidate in real time the step-by-step processes: from macrophage migration to the site of infection, to phagocytosis of mycobacteria, to migration of infected macrophages to deeper tissues in the lungs, to growth of mycobacteria within individual macrophages, to granuloma formation [70, 71].

Dendritic cells

It is now established that DCs are also involved in an effector role against M. tuberculosis infection [72, 73], and are central to the generation of acquired immunity after carriage of antigens to draining lymph nodes (LN), where recognition by T cells can be maximized [37, 38, 74]. To optimally prime pathogen-specific Th1 responses, DCs require stimulation through TLRs [75] by the pathogen as well as host-derived factors such as type I and type II IFNs, cytokines, and chemokines [76]. For example, M. tuberculosis dependent TLR2 ligation can promote the maturation of DCs via upregulation of costimulatory molecules and production of IL-12 essential to prime optimal Th1 responses [77]. However, ligation of DC-SIGN with the M. tuberculosis-derived LAM could lead to suppression of immune function

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through induction of IL-10 secretion [33, 43, 78]. In DCs, TLR9 signalling results in remodeling at the IL12p40 promoter leading to a robust IL-12 release [79].

The immune response limiting and switching off infection during primary TB is presumably initiated when, upon exposure to M. tuberculosis, the efficient antigen-capturing immature DCs [80], are transformed into mature T cell stimulating DCs, which migrate with high efficiency into draining LN. In these compartments, the stimulatory capacity of mature DCs ultimately leads to effector T cell differentiation and memory T cell expansion, which in turn, confer protection against M. tuberculosis in the lungs [81, 82]. In contrast to macrophages, DCs have poor mechanisms to eliminate internalized mycobacteria [83, 84]. Rather, it has been suggested that DCs offer a niche for long-term survival of intracellular bacteria [83, 84]. Thus, accumulation of DCs at the granuloma site during pulmonary infection with M. tuberculosis may provide niches where the bacteria can survive.

NK (T) cells

NK cells are a type of cytotoxic lymphocyte that is a major component of the innate immune system. These cells have been implicated in early immune responses to a variety of intracellular pathogens, including mycobacteria, through their capacity to rapidly produce IFN-γ and other immunoregulatory cytokines [85-87]. In mycobacterial infections, previous studies on the role of NK cells in host resistance have involved the use of antibodies that deplete NK populations. Although mice depleted of NK cells by this procedure were initially reported to be more susceptible to M. avium infection [88], this finding could not be reproduced in a later, more comprehensive study [89]. In contrast to these studies involving mice with an intact T cell compartment, severe combined immunodeficiency mice infected with M. avium were shown to be capable of forming hepatic granulomas, the response of which was demonstratedto be dependent on both IFN-γ and TNF [90]. More importantly, T cell receptor (TCR) αβ-deficient mice infected with M. tuberculosis were shown to survive longer than IFN-γ-deficient mice [91], a finding strongly suggestive of NK cell involvement. NK cells recruited to the lungs during mycobacterial infection are known to expand and become a primary source of IFN-γ [92]. In line with this, a protective role for NK-produced IFN-γ in T-independent host resistance to aerogenic M. tuberculosis infection has been demonstrated [93].

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Among the cell types that have been postulated to link the two arms of the immune system; the innate and adaptive immune responses, CD1d-restricted NKT cells are compelling candidates, being able to respond rapidly and subsequently to activate other cell types [94]. Because of their apparent self-reactivity and ability to quickly release large amounts of cytokines such as IFN-γ, NKT cells are hypothesized to be important in the initiation and regulation of various immune responses [94]. NKT cells are a subset of T cells that co-express an αβTCR, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. Unlike conventional αβ T cells, their TCRs are far more limited in diversity and recognize lipids and glycolipids presented by CD1d molecules, a member of the CD1 family of antigen presenting molecules, rather than peptide-MHC complexes. In mice, NKT cells are mainly represented by Vα14 NKT cells, while in humans, there is a homologous population of Vα24 NKT cells. A regulatory role for Vα14 NKT cells has been described during the course of mycobacterial infection in mice, through their ability to produce the anti-inflammatory cytokine IL-4, thereby limiting the extent of the inflammatory response [95]. This suggests an important role for this cell subset as a regulator of the balance between protective immune responses and immunopathology. Activated human CD1d-restricted NKT were capable of restricting the growth of M. tuberculosis in a granulolysin-dependent manner [96]. Furthermore, it has been shown that NKT cells induce a granulomatous response to a glycolipid fraction of M. tuberculosis cell wall [97].

Neutrophils

In infectious inflammation, polymorphonuclear cells, principally neutrophils are the first phagocytes to arrive from circulation and attempt to eliminate invading pathogens via oxygen-dependent and oxygen-independent mechanisms. The former mechanism results from the generation of reactive oxygen species [98], whereas the latter mechanism reflects the capacity of neutrophils to degranulate and release preformed oxidants and proteolytic enzymes from granules [99]. Neutrophils have been implicated in the control of mycobacterial infections [100, 101], but the mechanisms by which they exert direct protective functions are not completely resolved.

Some studies have demonstrated that human neutrophils are able to kill virulent M. tuberculosis [102], while others have not [103]. The recruitment of neutrophils to the lung has been described for acute TB in humans [102], and in experimental animals infected with

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mycobacteria [104]. A possible indirect role of neutrophils to mycobacteria killing was demonstrated by Tan and colleagues [105]. In this study, mycobacteria-infected macrophages acquired the contents of neutrophil granules and their antimicrobial molecules by the uptake of apoptotic neutrophil debris, which was trafficked to endosomes and co-localized with intracellular bacteria [105].

Both human and animal studies have shown that neutrophils may play an important role in the transition from innate to adaptive immune responses by producing critical cytokines and chemokines [106, 107].

γδ T cells

Human T cells expressing γδTCR represent a unique lymphocyte population with an unusual tissue distribution and antigen recognition pathway. Conditions that lead to responses of γδ T cells are not fully understood, and current concepts of γδ T cells as 'first line of defense', 'regulatory cells', or 'bridge between innate and adaptive responses' [108] only address facets of their complex behavior. The involvement of γδ T cells in the primary immune response to M. tuberculosis infection was described as early as 1989 [109]. Upon contact with mycobacteria, Vγ9/Vδ2 T cells have been shown to exhibit cytolytic functions and are hence involved in innate immune effector mechanisms [110, 111]. In these studies, the ability of Vγ9/Vδ2 T cells to kill mycobacteria was dependent on the release of preformed granules, perforin and granulysin [110, 111]. Murine studies have indicated that the induction of γδ T cells in the immune response against TB precedes that of conventional CD4 and CD8 cells, hence plays an important role in modulating the effector response against tuberculosis. For example, intranasal (i.n.) infection of mice with BCG resulted in an early accumulation of γδ T cells in the lungs, and the peak of γδ T cells expansion at 7 days postinfection preceded the 30 day peak of αβ T cells [112], suggesting that γδ T cells in the lungs might help to control mycobacterial infection before the onset of adaptive immunity.

Studies using γδ TCR knockout mice indicate that γδ T cells may be involved in the regulation of granuloma formation, which is critical for the control of mycobacteria [113]. Infection of mice deficient in γδ T cells with high dose M. tuberculosis resulted in the formation of pyogenic granulomas, suggesting that a role for these cells is perhaps in cellular traffic during mycobacterial infection [114]. In humans, loss of Vγ9+/Vδ2+ T cells, the major subset of the circulating γδ T cell pool correlated with pulmonary TB [115]. In mice, the

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reciprocal stimulation of γδ T cells and DCs was shown to be important for the optimal induction of antimycobacterial CD8 T cell response, indicating that stimulation of γδ T cells and their non-cognate interaction with DCs could be applied as an immune adjuvant strategy to optimize vaccine induced CD8 T cell immunity [116]. Recently, it has been shown that IL-17 production is dominated by γδ T cells rather than CD4 T cells during M. tuberculosis infection, thereby implicating them as a main player in the resistance against infection at the early stage [117].

Adaptive immunity

Several studies have shown that protective immunity to TB is dependent on the adaptive Th1 immune responses [61, 81, 118, 119], mediated by macrophages, DCs, T cells and their interactions, and depends on the interplay of cytokines produced by these cells [42, 61]. Clearance of bacteria by macrophages is in part dependent on macrophage activation by the cytokine IFN-γ secreted by CD4+ T cells, CD8+ T cells and NK cells [61, 81, 93, 118-120]. Infected macrophagessecrete pro-inflammatory cytokines such as TNF, IL-1 and IL-6,as well as chemokines that leads to the migration of monocyte derived macrophages and DCs to the site of infection [61, 37, 121]. The migration of cells to the site of infection results in the formation of granuloma, which functions to restrict further bacterial dissemination [122, 123]. The adaptive immune response is initiated when mycobacteria infected DCs mature and migrate to local LN, where recognition by T cells takes place [36, 37, 73, 74].

The hallmark of chronic infections such as TB is the significant delay between infection and the induction of the adaptive immune response, which allows early growth of the pathogen and the establishment of persistent infection. Recently, it was demonstrated that activation of M. tuberculosis-specific CD4+ T cells is dependent on trafficking of bacteria from the lung to local LN, and that delayed dissemination from the lung to sites of antigen presentation accounts for the lag in the initiation of adaptive immunity [38], (Figure 2) [124].

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Figure 2. Overview of the events that influence the initiation of the adaptive immune response after infection with M. tuberculosis (Immunol Cell Biology 2008; 86:293-294). Reprinted with permission from the Nature Publishing Group.

While the precise mechanisms for this delay are unclear, it has been suggested that low levels of antigen in early infection may help evade immune recognition and that some threshold level of antigen is required to stimulate the T-cell response [38, 125]. On the other hand, late migration of activated T cells to the lung was suggested to contribute to the delay in the onset of adaptive immunity [38].

The granulomatous response

The granulomatous response is a protective immunopathological response of the host following infection with M. tuberculosis. Pulmonary granuloma formation is a desperate attempt by the host immune system not only to contain multiplication and further dissemination of bacteria to other organs, but also to localize inflammation and prevent

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damage to the lungs. It is postulated that stimulated alveolar macrophages in the airways invade the lung epithelium following internalization of inhaled bacteria [122, 123]. Production of TNF and inflammatory chemokines from infected macrophages drive the recruitment of successive waves of neutrophils, NK cells, CD4+ T and CD8+ T cells, DC and B cells, each of which produces its own complement of cytokines that amplify cellular recruitment and remodeling of the infection site [90, 91, 122, 123, 126].

This inflammatory cascade is regulated and superceded by a specific cellular immune response that is linked to the production of IFN-γ. At this stage, formation of the 'stable' granuloma responsible for immune containment during latent or subclinical infection becomes recognizable and stratification of the structure emerges [127, 128]. The granuloma subsequently develops central areas of necrosis [129] (called caseum, from the word ‘cheese’ in Latin), resulting in the death of the majority of bacteria and destruction of the surrounding host tissue. The surviving bacilli exist in a latent state and can become reactivated leading to development of active disease. The granuloma serves 3 major purposes; it is a barrier to dissemination of bacteria throughout the lungs and other organs, a local environment in which immune cells can interact to kill bacteria, and a focus of inflammatory cells that prevent inflammation from occurring throughout the lungs [126].

The granuloma maintains a dynamic T cell population reflective of the systemic activated repertoire [123, 130], and are able to accumulate recently activated T cells. Disruption of the granuloma structure or function is therefore detrimental to the control of bacterial replication or immunopathology in the lung. In this regard, the reactivation of latent infection that stems from a failure of tissue granulomas to contain the organism has been reported in experimental models [61].

CD4+ T cells

In the majority of individuals exposed to M. tuberculosis, the innate response is not sufficient enough to protect against infection, and the adaptive immune response is necessary to restrict bacterial growth and mediate protection. Although various cells contribute to immunity against M. tuberculosis, T cells, notably effector CD4+ T cells play a dominant role [119, 131]. M. tuberculosis resides primarily in a vacuole within the macrophage, resulting in MHC II presentation of mycobacterial antigens to CD4+ T cells. Upon activation, CD4+ T cells

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secrete IFN-γ and TNF, which in turn induce antimycobacterial mechanisms in macrophages [61, 131, 132].

Studies in mouse models using antibody depletion of CD4+ T cells [133], adoptive transfer [134], or the use of gene-deficient mice [135], have demonstrated that CD4+ T cell subsets are required for the control of the infection. For example, CD4+ T cell-deficient mice infected with M. tuberculosis transiently displayed diminished levels of IFN-γ, yet succumbed to the infection [135]. In another study, depletion of CD4+ T cells resulted in reactivation of persistent TB in mice despite continued expression of both IFN-γ and NOS2 [136]. It has been demonstrated that CD4+ T cells are required for the development of a protective granulomatous response to pulmonary TB [137].

The important role of CD4+ T cells in the control of M. tuberculosis in humans is illustrated by the strong association of CD4+ T cell impairment and the reactivation of M. tuberculosis in patients with HIV infection [138]. Other roles played by CD4+ T cells include induction of apoptosis suggested to be important in controlling M. tuberculosis infection [139], conditioning of APCs, help for B cells and CD8+ T cells [140]. CD4+ T cells can also contribute to the control of acute mycobacterial infections through IFN-γ independent mechanisms, which have been demonstrated in experimental models using antibody depletion or mouse strains deficient in either CD4 or MHC class II molecules [136].

CD8+ T cells

Although mycobacteria reside within phagosomes, there is a large body of evidence that CD8+ T cells participate in immunity against M. tuberculosis infection [141]. It has been demonstrated that mycobacterial antigens derived from infected cells can be presented by MHC I to CD8+ T cells in both humans and mice, and antigens recognized by these cells have been identified [142]. CD8+ T cells also recognize various antigens from M. tuberculosis that are not presented by classical MHC I molecules, but by a closely related group of molecules, the Class Ib molecules. These are non-polymorphic, and include the CD1 molecules [reviewed in 143], as well as H2-M3. CD1 molecules primarily present lipid antigens from M. tuberculosis to CD8+ T cells, thereby increasing the possible antigen source greatly. Experimentally, mice deficient in β2-microglobulin, a component of both MHC I and non-classical MHC class Ib molecules were found to be more susceptible to infection with M.

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tuberculosis than wild type (WT) mice [144]. Moreover, increased susceptibility to mycobacterial infections has been observed in mice deficient in transporters associated with antigen processing, which transport peptides from the cytosol to the endoplasmic reticulum for loading into MHC I molecules [145, 146], indicating a protective role for CD8+ T cells.

In humans, CD8+ T cells can kill intracellular mycobacteria via the release of the antimicrobial peptide granulysin [147]; however, this molecule is not present in the mouse. The fact that no mouse analog of granulysin exists, may in part explain why CD8+ T cells are not as important in the control of infection in mouse models of TB [148]. The cytotoxic potential of CD8+ T cells to kill infected cells (Cytotoxic T cell; CTL activity) has been shown to be dependent on CD4+ T cells in the mouse model, suggesting that the susceptibility of CD4+ T cells knockout mice to M. tuberculosis infection might be due in part to impaired CTL activity [140]. CD8+ T cells also produce cytokines (IFN-γ and TNF) during M. tuberculosis infection, which probably participate in activation of macrophages [149].

B cells

Presently, there is a growing body of evidence demonstrating that B cells have a greater contribution to TB immunity than previously thought, and play a significant role in optimizing the host response against M. tuberculosis infection. For example, the identification of follicle-like B cell dominant structures within TB infected lungs of humans has suggested that B cells may play a previously unappreciated role in local immunity [150, 151]. Moreover, results from previous studies suggest that B cells influence the inflammatory progression in the lungs during M. tuberculosis infection [152, 153]. In mice, B cell-deficiency resulted in reduced recruitment of neutrophils, macrophages and CD8+ T cells to the lungs, suggesting a role for B cells in the regulation of chemokines and/or adhesion molecules [152]. A role for B cells in protection against M. tuberculosis infection was suggested on grounds of raised bacterial load in the organs of B cell-deficient mice [154]. An additional role for B cells as APCs has also been suggested [154].

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Soluble mediators of mycobacterial infections

Innate immune recognition of mycobacteria by phagocytic cells leads to cellular activation and rapid production of pro- and anti-inflammatory cytokines (Figure 3) [42]. These cytokines and chemokines recruit inflammatory cells (T cells, neutrophils and NK cells) to areas of infection, activate transmigrated cells, and coordinate the inflammatory and adaptive immune response to infection. The outcome of mycobacterial infections depends upon cytokine networks established and maintained by innate cells, of which macrophages are of critical importance. In addition to the well-defined cellular immune responses, individuals infected with mycobacteria mount a vigorous humoral immune response. In this thesis, the role of the following cytokines or cytokine receptors and antibodies in immunity to TB have been assessed, and will therefore be discussed; IFN-γ, IL-12, TNF, soluble TNF receptors (sTNFR) and antibodies.

Figure 3: Cellular immune response to M. tuberculosis (Immunological Reviews 2007; 219:167-186.). Reprinted with permission from Interscience-Wiley.

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IL-12

IL-12 is a product of phagocytic APCs, and acts as a pro-inflammatory cytokine that bridges the innate and adaptive immune responses and skews T cell reactivity toward a Th1 cytokine pattern [155, 156]. The bioactive IL-12p70 is a heterodimeric protein consisting of covalently linked p40 and p35 subunits, both of which are regulated independently [157]. IL-12 was the first cytokine to be described with potent Th1 promoting attributes, followed by IL-23 (shares the p40 component with IL-12), and more recently IL-27. Data from several studies indicate that the three cytokines together orchestrate Th1 responses, with IL-12 being the dominant cytokine that affects both the induction and maintenance of Th1 immunity [158]. Production of IL-12 by M. tuberculosis-infected DCs and macrophages is essential for the priming of potent Th1 responses, characterized by IFN-γ production by CD4+ and CD8+ T cells [61, 118, 119, 159, 160]. Humans with mutations in IL-12p40 or the IL-12 receptor genes have reduced capacity for IFN-γ production, and display increased susceptibility to environmental mycobacteria and BCG [161]. Moreover, a role for IL-12 in resistance to M. tuberculosis was suggested by the improved clinical outcome observed when the cytokine was combined with drug therapy in a case study [162].

In mice, early administration of IL-12 after M. tuberculosis infection resulted in a significantly decreased bacterial burden, and increased mean host survival time [163]. Moreover, neutralization of IL-12 at the initiation of M. tuberculosis infection led to increased bacterial loads and reduced granuloma integrity [162]. Furthermore, mice deficient in IL-12p40 were highly susceptible to M. tuberculosis infection [164]. It has been shown that the administration of IL-12 could substantially reduce bacterial numbers in mice with a chronic M. tuberculosis infection [165], suggesting that the induction of this cytokine is an important factor in the design of TB vaccines.

IFN-γ

IFN-γ produced mainly by CD4+, CD8+ T cells, NK and γδ T cells during M. tuberculosis infection [61, 93, 118, 119, 131, 166] is critical for macrophage activation and the subsequent induction of microbicidal mechanisms. Individuals defective in genes for IFN-γ or IFN-γ receptors are susceptible to serious mycobacterial infections, including M. tuberculosis [161]. In a large study, it was reported that patients with IFN-γ receptor-deficiency developed

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disseminated infection with M. bovis BCG or environmental mycobacteria, which in some cases resulted in death of about half of the patients and required continuous antimycobacterial treatment in the survivors [reviewed in 161]. In mice, IFN-γ knockout strains are the most susceptible to virulent M. tuberculosis infection [167], with defective macrophage activation and low NOS2 expression [167-169].

M. tuberculosis has developed mechanisms to limit the activation of macrophages by IFN-γ [21-30], suggesting that the amount of IFN-γ produced by T cells may be less predictive of outcome than the ability of the cells to respond to this cytokine. In this regard, it has been shown that the level of IFN-γ produced by a mouse in response to a candidate vaccine does not always correlate with the effectiveness of the vaccine during M. tuberculosis challenge [170]. Similarly, evaluation of the efficacy of human BCG vaccination using several assays demonstrated that mycobacterial growth inhibition did not correlate with IFN-γ response [171]. Thus, although IFN-γ is essential for the development of an immune response that prolongs the life span of an infected animal, it is not sufficient to eliminate an M. tuberculosis infection.

TNF and soluble TNF Receptors

TNF is produced primarily by activated monocytes/macrophages in response to pathogens, but can also be expressed by activated T cells, B cells, NK cells, and some tumor cells [172]. TNF is first synthesized as a transmembrane (TmTNF) precursor and cleaved by a membrane-bound metalloprotease disintegrin, TNF converting enzyme (TACE), generating a soluble TNF molecule [173]. Both forms of TNF function physiologically by interacting with one of two receptors; TNFR1 (55 kDa) and TNFR2 (75 kDa) expressed on a diverse range of cell types [172]. Upon stimulation, these receptors could be cleaved from the cell surface, or directly expressed as soluble isoforms lacking the transmembrane domain. TNF mainly binds to TNFR1 while the TmTNF binds to TNFR2 [174, 175].

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Figure 4: Schematic representation of the central role of TNF in the cellular immune response to M. tuberculosis infection (Lancet Infect Dis 2003; 3:148-155). Reprinted with permission from Elsevier.

The importance of TNF in the generation and maintenance of a protective immune response against M. tuberculosis, and other bacterial and viral pathogens has been clearly demonstrated [176-178]. Although TNF is not required for the generation of antigen-specific T cell responses, it is essential for controlling the recruitment of inflammatory cells to sites of infection and the development of a protective granulomatous response, resulting in containment of bacillar growth and survival of the infected animals [126, 178-180]. During M. tuberculosis infection, TNF is involved in almost every stage of the inflammatory response, from the initial macrophage response, to the attachment, migration, and trafficking of leukocytes through blood vessels, to retention at the site of infection and in immunopathology (Figure 4) [Reviewed in 181]. For example in TNF-knockout mice, the inflammatory response generated following M. tuberculosis infection was highly

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dysregulated, and T cells recruited to the lungs failed to migrate into central lesions,thereby limiting their contact with infected macrophages [182]. In addition, mice deficient in TNF or sTNFR1 succumbed quickly to M. tuberculosis infection, with substantially higher bacterial burden compared to their WT counterparts [176].

TNFR1 signalling is required for the modulation of T-cell responses, because in TNFR1-deficient mice, T-cell dependant granuloma decomposition is observed [180], whereas TNFR2 seems to have a lesser role in granuloma formation and mycobacterial immunity. Although soluble TNF is required for the long term control of M. tuberculosis infection, TmTNF was sufficient to control acute, but not chronic infection [183, 184]. However, sTNFR neutralization of TNF is important for homeostasis, since excessive production could lead to exaggerated inflammation resulting in tissue damage.

Antibodies

Historically, the view that protective immunity against TB is mediated exclusively by T cells, involving cytokines, mainly IFN-γ-mediated activation of infected macrophages, rather than antibodies has determined all strategies of TB vaccine research. This view has been sustained by the notion that antibodies cannot reach the bacilli within the phagosomes of infected macrophages [185], and the initial difficulty in demonstrating a consistent protective effect of antibodies in M. tuberculosis infection [186]. However, the fact that TB develops despite the presence of abundant T helper immunity [187], coupled with the observation that T-cell targeted vaccination does not always induce optimal protection either in humans or in experimental animals, has made it necessary to investigate alternative immune mechanisms of protection [188].

To this end, the protective role of antibodies in TB has been elucidated recently using modern approaches and tools [reviewed in 189, 190]. IgA is the primary immunoglobulin isotype induced at mucosal sites [191], and is thought to mediate defense functions at these sites [192]. Secretory IgA in mucosal secretions has been shown to prevent the adsorption of pathogens and to neutralize their toxic products at the mucosal epithelium [192]. With regard to TB, previous studies in our group demonstrated that mice deficient in IgA [193], or the polymeric Ig receptor [194], and thereby incapable of actively transporting either IgA or IgM, were more susceptible to mycobacterial infection than their WT counterparts. Furthermore,

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the beneficial effect of passively administered IgA or IgA in synergy with IFN-γ on the survival of animals infected with mycobacteria has been demonstrated [185, 188, 195]. In addition, the possible role of antibodies in humans to the natural course of M. tuberculosis infection was indicated in clinical studies, which reported higher antibody titres to Ag85 in patients with milder forms of active TB [196].

Mucosal immunity in pulmonary TB

Mucosal immunization has received increasing attention because the respiratory tract is the natural route of M. tuberculosis infection,and it is believed that mucosal vaccination provides the bestprotection from mucosal infectious diseases [197]. Emerging evidence suggests that respiratory mucosal vaccination provides better immune protection against pulmonary TB than parenteral vaccination [198, 199]. For example, respiratory mucosal immunization uniquely elicited higher numbers of antigen-specific CD4+ and CD8+ T cells in the airways capable of IFN-γ production,cytotoxic lysis of target cells, and immune protection againstM. tuberculosis infection. In comparison, parenteral intramuscular (i.m.) immunization led to activation of T cells, particularly CD8+ T cells, in theperipheral lymphoid organs, but failed to elicit airway luminal T cells or protect the lung from M. tuberculosis infection [200]. Furthermore, airway exposure to an otherwise non-immunogenic soluble M. tuberculosis antigens resulted in recruitment and retention of antigen-specific T cells in the airway lumen, which were capable of robust protection against pulmonary M. tuberculosis challenge [201]. It has been proposed that the failure or success of parenteral immunization hinges critically on T cell geography, whether antigen-specific T cells are within or outside of the mucosal lumen at the right time in order for immune protection to occur [201]. The immunoprotective role of mucosally induced IgA against mycobacterial infection has been demonstrated [193, 202].

The lungs are the site of primary exposure to several pathogenic microorganisms, including mycobacteria, and local immunoregulatory mechanisms are essential to ensure that immune effector mechanisms remain quiescent or are activated as necessary. The major lung accessory cells with immunoregulatory capacity are alveolar macrophages [203] and pulmonary DCs [204]. Alveolar macrophages are professional phagocytes residing within the alveoli and capable of rapidly clearing large numbers of bacteria in the lung when activated [61, 205].

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DIAGNOSIS OF TB

The most powerful tool in any TB control program is prompt diagnosis and successful treatment of patients with active contagious disease. In this regard, existing tests for diagnosis of TB vary in sensitivity, specificity, speed and cost.

Sputum smear microscopy

The use of stained-sputum microscopy (Ziehl-Neelsen, Kinyoun, or fluorochrome) for acid-fast bacilli still remains the most available, easy to perform, inexpensive, and rapid diagnostic test for TB [206]. This is especially true for laboratories in developing countries [207], where there are limited resources. The greatest difficulty in diagnosing TB and other mycobacterial infections by sputum microscopy is the test’s sometimes lack of sensitivity and specificity [208]. Further, diagnosis of TB by microscopy is difficult especially in children who rarely produce adequate sputum. Currently, the sensitivity of this test has improved considerably with improved techniques and standardization of sputum preparation, and the use of auramine-rhodamine/fluorochrome method instead of the classic Ziehl-Neelsen stain which uses carbol-fuchsin [209]. Identification of smear positive patients is of major importance, because only smear positive pulmonary TB patients are regarded as highly infectious to others [210].

Bacteria cultivation

Mycobacterial culture is the ultimate proof of mycobacterial infection and is often used as a reference method due to its high sensitivity and specificity [211, 212]. However, it takes 4-6 weeks for M. tuberculosis to grow on solid culture medium (e.g. agar based Middlebrook 7H10 or 7H11 or the egg-based Lowenstein-Jensson medium), and 3 weeks to grow in liquid 7H9 medium [213]. Notwithstanding the long culture period, it is still a requirement for definitive diagnosis of TB and in drug-susceptibility testing [214].

Biomarkers

A biological marker (biomarker) is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or

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pharmacological responses to a therapeutic intervention [215]. For example with regard to treatment, a good biomarker must measure a factor that is part of the pathological process leading to the clinical endpoint and should be scientifically plausible. TB diagnostic tests that rely on detection of host immunological markers currently in use include the tuberculin skin test (TST) [216, 217] and interferon gamma release assays (IGRAs) [218, 219]. TB infection is controlled by cell-mediated immunity, and the reactivity of sensitized lymphocytes in vivo (TST), or IFN-γ release in vitro are expected to be strong indicators of exposure, disease progression or treatment.

Tuberculin skin test

The TST or Mantoux test or purified protein derivative (PPD) test has been used for almost a century as the standard test for the diagnosis TB infection and disease [220]. The TST test is based upon the type 4 hypersensitivity reaction, in which a standard dose of 5 Tuberculin units is injected intradermally into the forearm and read 48 to 72 hours later [221]. The TST is based on the principle that T cells of individuals sensitized with mycobacterial antigens produce IFN-γ when they re-encounter these antigens (Figure 5) [218]. The reaction is read by measuring the diameter of induration across the forearm, perpendicular to the long axis in millimeters. No induration is recorded as "0 mm", whereas reactions over 10 mm in size are considered positive in non-immunocompromised persons. The main drawback with the clinical use of the TST is the lack of specificity due to cross-reactivity with proteins present in other mycobacteria, such as BCG or mycobacterium other than tuberculosis (MOTT) [220, 221]. Moreover, several factors may contribute to false-negative results such as age, poor nutrition, acute illness or immunosuppression induced by medication or HIV infection [210].

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Figure 5. Biological basis of the TST and IFN-γ release assays. (Lancet 2000; 356:1099-1104). Reprinted with permission from Elsevier.

Interferon gamma release assays

As a replacement for the Mantoux test, several other tests are being developed. IGRAs are based on the same principle as the TST, that T cells of individuals sensitized with M. tuberculosis produce IFN-γ when they re-encounter mycobacterial antigens (Figure 5). IGRAs quantify the amounts of antigen-specific IFN-γ in blood culture supernatants (QuantiFERON-TB Gold, Cellestis Limited, Carnegie, Victoria, Australia) or determine the frequencyof IFN-γ producing blood leukocytes T SPOT-TB assay (Oxford Immunotec, Oxford, UK) in response to specific mycobacterialpeptides [222]. These newer assays use antigens specific to M. tuberculosis, such as the early secretory antigenic target 6 (ESAT-6) and culture filtrate protein (CFP) 10.

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These proteins are encoded by genes located within the region of difference 1 of the M. tuberculosis genome, and are not shared with any BCG substrains or most MOTT species, with the exception of M. kansasii, M. marinum, M. flavesence and M. szulgai [218, 223]. The test is used in conjunction with risk assessment, radiography and other medical and diagnostic assays. However, while IGRAs offer comparably high sensitivities and specificities in the diagnosis of TB in immunocompetent patients, there is concern about the sensitivity in immunocompromised patients, especially when using the QFT-TB test [224, 225]. Thus, IFN-γ based assays may give false negative TB diagnosis in endemic areas with high burden of HIV coinfection where reliable diagnostic tools are needed the most.

Serodiagnosis

Mycobacterial infections elicit the production of antibodies to several antigens that may be used as markers of TB infection. Sero-diagnostic tests are usually simple and rapid, and do not involve the use of living cells or direct detection of bacteria in specimens. Different mycobacterial preparations have been evaluated as candidates for the development of serodiagnostic assays, including CFPs [226], purified extracts of glycolipids [227], mycobacterial sonicates [228] and PPD [229]. In vitro cultivation of M. tuberculosis results in the accumulation of proteins in the extracellular milieu, collectively termed CFPs. By virtue of their immunodominant nature [230], and capacity to activate both humoral and cell-mediated immune responses, these CFPs appear to be the most promising for proteins for use in the diagnosis of TB. Some important CFPs are the 6-kDa antigen or ESAT-6, CFP-10, the 19-kDa, the 30- to 31-kDa Ag85 complex (Ag 85) and the 38-kDa.

A major fraction of the secreted proteins in M. tuberculosis culture filtrate is formed by the Ag85, a family of proteins (Ag85A, Ag85B and Ag85C) [231]. The potential use of Ag85 for TB diagnosis has been evaluated by many investigators with varying sensitivities or specificities [232, 233]. The 38-kDa antigen is a surface expressed glycolipoprotein, and is one of the most important antigens used in the development of serodiagnostic tests [234]. In an enzyme-linked immunosorbent assay (ELISA) format, up to 85% of smear-positive cases were detected [235]. However, as a single antigen, the 38-kDa antigen may lack sufficient sensitivity to create an optimal serodiagnostic test, especially for smear-negative individuals, where sensitivity is considerably lower [235]. The 19-kDa has been shown to be recognized

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by sera from TB patients [235, 236]. Varying sensitivities have been demonstrated, which corresponded with the disease pathology [236].

The 16-kDa heat shock protein X, is specific to the M. tuberculosis complex [237], and essential for mycobacterial persistence within macrophages. It is the dominant protein produced during static growth or under oxygen deprivation [238]. The 16-kDa antigen has been used for the detection of antibody isotypes in the sera of TB patients alone, or in combination with other antigens [239-242]. In addition, both T-cell and B-cell responses to the 16-kDa antigen were found to be associated with latent M. tuberculosis infection [243, 244], pointing to the importance of 16-kDa as an antigenic target of immune responses during latent TB infection. This antigen has been incorporated into a commercial kit in combination with the 38-kDa antigen (Pathozyme TB complex, Omega Diagnostics Ltd, Alloa, Scotland).

Molecular methods

Nucleic acid amplification tests, such as polymerase chain reaction have contributed to a more rapid and reliable diagnosis of pulmonary TB. These technologies allow for the amplification of specific target sequences of nucleic acids that can be detected through the use of nucleic acid probes; both RNA and DNA amplification systems are commercially available [245, 246]. Amplification methods for M. tuberculosis, however, have low sensitivity, and the absence of specific internal controls for the detection of inhibitors of the reaction means it cannot completely replace the classical diagnostic techniques [246].

THE BCG VACCINE

M. bovis Bacillus Calmette-Guérin (BCG) is the most widely used vaccine in the world, and approximately three billion people have been vaccinated since 1921. Close to 115 million doses are distributed each year [247, 248], providing almost 80% coverage of infants worldwide. Robert Koch (1843-1910) elucidated the etiology of TB, and Calmette (1863-1933), together with Guérin (1872-1961), developed the BCG vaccine in the 20th century, which is still the only vaccine available against TB. The first clinical studies took place from 1921 to 1927 in France and Belgium, and showed that BCG was highly efficient in protecting against TB in children [249]. Unfortunately, despite the early success, the BCG vaccine has

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had a limited effect against the TB epidemic in developing countries [250]. Although BCG protects children efficiently against the early manifestations of TB, it hardly offers protection against adult pulmonary TB [251].

The reasons for the varying efficacy of BCG in protection against pulmonary TB are not completely understood. However, potential explanations that have been suggested include: interference with the immune response to BCG caused by previous exposure to environmental mycobacteria [252]; differences among BCG vaccine sub-strains; phenotypic changes in the vaccine during passage from the original cultures and during the manufacturing process. Other factors are; the deletion of protective antigens from BCG; failure of BCG to stimulate adequate, balanced antimycobacterial CD4+ and CD8+ T-cell responses; variability in dose, and route of administration [247]. The current route of vaccination, the intradermal route is thought of as not inducing an optimal immune response. Vaccination via the respiratory tract is believed to be superior to vaccination at other sites in conferring protection against several mucosal infections [253]. In line with this, i.n. vaccination with BCG led to better protection of mice against challenge by M. tuberculosis [202] or M. bovis [254], which was attributed to an enhanced and more rapid production of IFN-γ by T cells [198]. Also, better protection was observed in the majority of studies using aerosol delivery of BCG [255]. Apart from the immunological advantages offered by i.n. vaccination, there are logistic advantages such as vaccination without needles and syringes, which will make immunization more acceptable, safer and better suited for mass administration.

Development of New TB vaccines

Substantial efforts are currently being put into the development of new TB vaccines to either replace or boost the existing BCG vaccine, albeit the fact that the BCG vaccine provides varying degrees of protection against TB. Among the spectrum of innovative new approaches that have been applied to TB vaccine development during the last decade, some have relied on strengthening the immunogenicity and/or persistence of genetically modified recombinant BCG strains [256], and others on using attenuated mycobacteria such as auxotrophic M. tuberculosis strains [257, 258] or less virulent mycobacteria, such as M. microti, M. vaccae, or M. smegmatis that overproduce immunogenic antigens of M. tuberculosis [259, 260]. Re-engineering BCG is an interesting approach that relies on the basic premise that the efficacy of BCG could be enhanced through insertion of genes encoding immunodominant antigens or

Mycobacterial infection: Immune evasion, host susceptibility and immunological markers of diagnostic importance